The wealth of information made available through efforts in structural genomics and advances in computation has allowed structure-based drug design to emerge as a valuable tool in medicinal chemistry. In the past combinatorial chemistry, coupled with high-throughput approaches, shifted attention away from the more structure-based methods. Large-scale determination of protein structures is reversing the drug discovery process by starting with the protein structure and using it to identify and design new ligands. It is the integration of structure-based methods, virtual screening, and combinatorial chemistry that will provide the basis for more efficient drug design in the future, significantly reducing the time of the design cycle and the cost per marketed drug. Significant advances have already been made in AIDS, arthritis and cancer and in the treatment of hypertension (e.g. captopril).
Translation of the genetic code occurs on the ribosome, a large nucleoprotein complex that consists of two subunits. In bacteria, the two subunits are denoted 30S and 50S. The 50S subunit contains the catalytic site of peptidyl transferase activity, while the 30S subunit plays a crucial role in decoding messenger RNA. Protein synthesis is a complex, multistep process that requires several extrinsic GTP-hydrolysing protein factors during each of the main stages of initiation, elongation and termination. Despite several decades of work, the molecular details of the process are poorly understood, and the elucidation of the mechanism of translation is one of the fundamental problems in molecular biology today. A recent collection of articles summarizes the state of understanding of the field [1].
A contribution to this problem was made by Yonath and coworkers, who after nearly a decade of work showed that structures as large as the 50S ribosomal subunit would form crystals that diffract beyond 3 Å resolution [2]. Originally, it was not clear that phase information from such a large asymmetric unit could be obtained to high resolution, but the development of bright, tuneable synchrotron radiation sources, large and accurate area detectors, vastly improved crystallographic computing, and the advent of cryocrystallography have all contributed to making structural studies of the ribosome more tractable. In our work, the use of anomalous scattering from the LIII edges of lanthanides and osmium has also played a critical role in obtaining phases.
The 30S ribosomal subunit (hereafter referred to as 30S) from Thermus thermophilus was originally crystallized by Trakhanov et al. in 2-methyl-2,4-pentanediol (MPD) [3] and soon afterwards by Yonath and coworkers in a mixture of ethyl-butanol and ethanol [4]. Subsequent work by both groups showed that the MPD crystal form diffracted to about 9-12 Å resolution [5, 6]. The diffraction limit of these crystals did not improve beyond 7 Å resolution for almost a decade, but more recently both Yonath and coworkers [7, 8] and we [9] obtained crystals of the MPD form that exhibit significantly improved diffraction. However, unlike the crystals obtained by the Yonath group [6], our crystals do not require soaking in tungsten clusters or heat treatment in order to obtain high resolution diffraction.
We have previously described the structure of the 30S at 5.5 Å resolution [9]. We were able to place all seven proteins whose structures were known at the time, infer the structure of protein S20 to be a three-helix bundle, trace the fold of an entire domain of 16S RNA, and identify a long RNA helix at the interface that contains the decoding site of the 30S. Proteins S5 and S7 were also placed in electron density maps of the 30S obtained by Yonath and coworkers.
The 30S ribosomal subunit is a major target for antibiotics. The ribosome is a useful target for antibiotics since the structure of the 30S is widely conserved between prokaryotes, allowing for broad spectrum antibiotics. However, resistance to current antibiotics is currently a major problem in the field of medicine. There are presently very few new antibiotics available which can be used to treat the highly resistant strains of bacteria such as MRSA (methicilin resistant Staphylococcus aureus) which are becoming increasingly widespread.
Understanding the interaction of antibiotics with the ribosome at the molecular level is important for two reasons. Firstly, antibiotics act by interfering with various aspects of ribosome function. Thus understanding their interaction will help shed light on mechanisms involved in translation. Secondly, a detailed knowledge of antibiotic interactions with the ribosome could aid the development of new drugs against increasingly resistant strains of bacteria. Although antibiotics were characterized several decades ago, a detailed knowledge of their mechanism will in general require a three-dimensional structure of their complex with the ribosome.
The low (greater than 3 Å) resolution crystal structures described above do not provide sufficiently detailed resolution for useful modelling of the crystal structure of the 30S and there is thus a need for a high resolution structure which can be useful in the development of novel therapeutics.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.